Previously, we identified b2 Off cone bipolar cells receiving direct rod inputs as an alternative rod pathway. We studied the kinetics of the rod synapse by monitoring the postsynaptic responses from Off cone bipolar cells, unfiltered by mGluR6. We found that the rates of vesicle release and replenish are similar in rods and cones. Here we study the function of this alternative rod pathway. 1. The classic rod pathway exists in the cone dominated ground squirrel retina. One could argue that the rod pathways in this cone dominant mammal are abnormal and connections between rods and b2 cells might result from the absence of a canonical rod pathway. We identified a RBC-like cell type that has axon terminals that ramify at the bottom of the inner plexiform layer (IPL) and is labeled by an antibody against the RBC marker PKC-alpha. An antibody against calretinin that labels AII amacrine cells in primate and rabbit retinas, also labeled an AIIlike amacrine cell in the ground squirrel. The dendrites of calretinin-positive amacrine cells express connexin 36 a hallmark of AII amacrine cells in the mammalian retina. Hence, by anatomical criteria, the ground squirrel retina does contain the neuronal components of the classic rod pathway. 2. Rod signal through rod-cone coupling can be detected in b2 bipolar cells. We observed electrical coupling between ground squirrel rods and cones, and identified a slow component of transmission between rods and b2 cells that was most likely due to rod-cone coupling. The rod-cone junctional conductance measured by paired-whole cell recordings was comparable to that previously reported for cone-cone pairs (121 80 pS, n = 9). Because of the predominance of cones, it was easier to study the mechanism of the delayed EPSC peak by depolarizing a cone and recording from a b2 cell (similar results were obtained at rod to b2 cell synapses). We stepped a cone from a holding potential of -70 mV to a series of voltages between 50 and 90 mV and measured the EPSC in a b2 bipolar cell. The peak of the initial b2 EPSC transient closely tracked the peak cone Ca2+ current. The amplitude of the secondary b2 EPSC response, however, increased monotonically with cone voltage, and was most prominent even when the first component, and thus release from the depolarized cone, is absent or ran down. A straightforward interpretation is that the initial b2 cell response is produced by a direct synaptic contact, and decreases when the driving force for Ca2+ influx is reduced at depolarized membrane potentials. The broad second component, may involve release from neighboring cones that are activated via electrical synapses. Indeed, a typical rod step depolarization from 70 to 30 mV produced a 5 mV depolarization in adjacent cones, which should activate the cone Ca2+ channels and trigger transmitter release. 3. The direct synapse between rods and Off CBCs may mediate fast rod signaling. We compared the speed of transmission following a step depolarization at the rod to Off b2 cell synapse, the rod-cone electrical synapse, and at a cone to On bipolar cell synapse (cone-On CBC responses were analyzed, which should have similar kinetics as rod-RBC responses, given that they have the same type of postsynaptic receptor mGluR6). Signals in the rod to b2 cell pathway rose and reached their peak 5-10 times faster than the signals in the other pathways. By comparing the time-to-peak of photoreceptor-b2 EPSC and photoreceptor-ganglion cell EPSC, we found that the delay between b2 EPSC and the EPSC in the downstream ganglion cell appeared to be minimal (3 ms). Therefore, the speed of rod signal transmission is primarily determined by how fast signals reach CBCs. Thus the rod-b2 bipolar cell pathway may mediate fast rod signaling. Summery and Significance: This study provided the first direct evidence that contacts between rods and Off CBCs are functional synapses. It revealed properties of the mammalian rod synapse that, until now, have remained elusive. It also included the first direct measurement of rod-cone coupling in the mammalian retina and identified the bipolar cell responses to rod signals transmitted through rod-cone gap junctions. Finally, it identified a fast rod signaling pathway that is important for rod vision in higher light levels. In sum, the project greatly extended our knowledge about the first synapse in rod vision and multiple pathways carrying rod signals.